The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. Genome-wide gene expression profiling carried out in an independent project, combined with a survey of the literature, uncovered numerous transcription factors (TFs) that are expressed in various subsets of mesodermal cells at different developmental stages. For many of these TFs, prior genetic studies have implicated an important role in muscle and/or heart development. However, in most cases, the DNA binding specificities and regulatory targets of these TFs have not been characterized. To address this critical knowledge gap, we have initiated a systematic analysis of the sequences bound by mesodermal TFs using the protein binding microarray (PBM) technology developed by our collaborators in Dr. Martha Bulyks laboratory at Brigham and Womens Hospital and Harvard Medical School. To date, we have completed PBM analysis of over 40 Drosophila mesodermal TFs. Our most comprehensive PBM dataset for a single family of TFs corresponds to a group of Drosophila homeodomain (HD) proteins that are localized to the mesoderm. Extensive genetic evidence has established that certain HD TFs serve as key determinants of muscle founder cell (FC) identity during Drosophila embryonic myogenesis. However, since HDs, in general, are known to bind to related and highly overlapping DNA sequences, the molecular basis of the developmental specificity of FC identity HD (FCI-HD) proteins remains unknown. As an initial step in addressing this problem, we used universal PBMs to interrogate all possible binding site sequences of a number of FCI-HDs, including Slouch (Slou) and Muscle segement homeobox (Msh). These experiments revealed the complete spectrum of 9-mers to which Slou and Msh bind. Detailed analyses of these results indicated binding sites characterized by a TAAT core that are shared by both proteins, as well as a small number of unique motifs that deviate from this consensus sequence and which are preferentially recognized by one but not by the other FCI-HD. Other HDs were similarly found to have distinct subsets of unique binding motifs. During the past year, we have evaluated the functional significance of these HD-preferred recognition sequences in an independent project. In the case of TF families containing additional classes of DNA binding domains, we have used available PBM data from other species to infer the DNA binding specificities of orthologous Drosophila TFs in order to design functional studies of gene regulatory models. For example, this approach proved critical to an independent project in which we established that cell type-specific Forkhead TFs differentially regulate the activity of a single enhancer that is active in multiple mesodermal derivatives by binding to different sites that possess two unique DNA binding specificities. In yet another related project, incorporation of DNA binding data derived from other published sources provided critical information for the computational classification of muscle FC enhancers and the prediction and subsequent experimental validation of novel motifs that contribute to the combinatorial specificity of muscle gene regulation. Collectively, these investigations serve as a paradigm for how in-depth knowledge of TF binding sites can be utilized to dissect the molecular mechanisms responsible for generating the unique genetic programs of individual cells during development.